To 12-0-tetradecanoyl-phorbol-13-acetate Research Articles

Biological and technical variables in myc expression in HL60 cells exposed to 60 Hz electromagnetic fields

The purpose of the present report is two-fold: first, to find an explanation for the inability of two groups of investigators, Lacy-Hulbert et al. and Saffer and Thurston [A. Lacy-Hulbert, R. Wilkins, T.R. Hesketh, J.C. Metcalfe, No effect of 60Hz electromagnetic fields on MYC or β-actin expression in human leukemic cells, Radiation Res. 144 (1995) 9–17; J.D. Saffer, S.J. Thurston, Short exposures to 60 Hz magnetic fields do not alter MYC expression in HL60 cells or Daudi cells, Radiation Res. 144 (1995) [18–25], to replicate our results, and second, to examine the broader issues involved in in vitro studies and replication in bioelectromagnetics. Replication experiments mandate that the precise cell type population be used and that the experimental protocol (i.e., exposure system, conditions of exposure, techniques for extraction and measurement) be followed exactly. By this criterion, the reported experiments of Lacy-Hulbert et al. and Saffer and Thurston were not replications. In this paper, we present replication experiments that again show a significant increase in c- myc transcript levels, although at a lower level. We also introduce variations from our protocol that were used in the reported ‘replication’ experiments. A major departure from the original experiments was use of different populations of HL60 cells; American Type Culture Collection (ATCC) as opposed to the original Columbia University Cancer Center (CUCC). The growth characteristics and responses to 12-0-tetra-decanoylphorbol-13-acetate (TPA) of the two cell populations showed significantly different reactivities. In line with these characteristics, c- myc increased in CUCC cells and did not increase in ATCC cells when stimulated by EM fields. We also compared other differences in protocol: SDS/phenol and guanidine/phenol RNA extraction procedures, the stability/variability of two housekeeping genes, β-2 μglobulin and GaPDH, as internal standards, sham-exposed controls versus inactive coil and double-wound coils versus single-wound coils. In all experiments, cell were shielded within mu metal containers during exposures. The shielding in one of the ‘replication’ studies, Lacy-Hulbert et al.'s, suggest that both experimental and control cells, which were side by side in the apparatus, were exposed to EM fields.

Phenobarbital-Induced Activation ofCYP2H1and 5-Aminolevulinate Synthase Genes in Chick Embryo Hepatocytes Is Blocked by an Inhibitor of Protein Phosphorylation

The phenobarbital-induced activation of cytochrome P4502H1 (CYP2H1) and 5-aminolevulinate synthase (ALAS-1) genes in chick embryo hepatocytes occurs at the level of gene transcription, but the molecular mechanism underlying this induction is not understood in detail. In the present study, we report that the protein kinase inhibitor 2-aminopurine markedly inhibits the phenobarbital-induced activation ofCYP2H1and ALAS-1 genes as measured by Northern blot analysis, but does not alter the basal expression of these genes in the absence of drug. Transient expression studies confirmed these findings. The construct pCATBg4.8 contains a 4.8-kb drug-responsive domain of theCYP2H1gene fused to the enhancerless SV40 promoter and the drug-induced expression of this construct in chick embryo hepatocytes was inhibited by 2-aminopurine. Another construct pCAT, with the first 547 bp of 5′ flanking region of theCYP2H1gene, is not responsive to drug and basal expression of this construct was not altered by the addition of 2-aminopurine. The evidence presented here demonstrates that the inhibitory action of 2-aminopurine on drug-induction is not due to a toxic effect on the cells. The induction of theCYP2H1gene by phenobarbital was not altered by treating cells with the specific inhibitors for protein kinase C (GF 109203X and Ro 31-8220) or prolonged exposure to 12-0-tetradecanoyl- phorbol 13-acetate (TPA) or treatment with the specific inhibitors for tyrosine kinase (genistein and tyrphostin A25). Overall, the data indicate that a 2-aminopurine-sensitive protein kinase activity is required for the phenobarbital-induction mechanism but this is unlikely to be a protein kinase C or tyrosine kinase. It can be postulated that phosphorylation of a drug receptor protein may be an important step in the drug-induction process.

The effect of differentiation inducers on the integrin expression of K562 erythroleukemia cells.

We studied the effects of differentiation-inducers on the integrin profile and adhesive properties of K562 leukemia cells. The fibronectin (Fn) receptor integrin, alpha 5 beta 1, was the only integrin expressed in suspension cultured K562 cells. When the cells were exposed to 12-0-tetradecanoylphorbol-13-acetate (TPA) immunoreactivity for the beta 1 integrin subunit was slightly enhanced. TPA exposure also induced the appearance of the alpha 2, alpha 3, alpha v and beta 3 integrin subunits, but the platelet integrin subunit alpha IIb was not detected. On the other hand, hemin chloride-induced erythroid differentiation of K562 cells diminished the expression of the alpha 5 beta 1 integrin on the surface of the cells. Adhesion experiments with TPA-exposed K562 cells indicated that although the adherence to the extracellular matrix (ECM) proteins as a rule was low a few cells spread on these proteins. The present results specify the effects of differentiation inducers on the integrin profile of K562 cells and excludes the comprehension that TPA would induce expression of the platelet integrin alpha IIb on their surface. Our results also show, that an increased expression of a certain integrin does not necessarily lead to a comparable adhesion ability on its ligand in vitro.

Rapid Inactivation and Phosphorylation of Pyroglutamyl Peptidase II in Y-79 Human Retinoblastoma Cells after Exposure to Phorbol Ester*

Pyroglutamyl peptidase II (EC 3.4.19.-), a membrane-bound metalloproteinase, is a highly specific TRH-degrading enzyme. Exposure of Y-79 human retinoblastoma cells to 12-0-tetradecanoyl phorbol 13-acetate (TPA) decreased the activity of this enzyme in a time- and concentration-dependent manner (IC50 5 x 10(-9) M). After 15 min of TPA treatment, only 10% of pyroglutamyl peptidase II activity remained. TPA treatment did not affect the activity of the cytosolic enzyme pyroglutamyl peptidase I (EC 3.4.19.3) or the membrane-bound enzyme dipeptidyl peptidase IV (EC 3.4.19.3). Pretreatment of the cells with the protein kinase C inhibitors H-7 or sphingosine prevented the inactivation of pyroglutamyl peptidase II by TPA. The time course of the TPA-mediated effect paralleled the time course of translocation and activation of protein kinase C in this cell line. Immunoblot analysis demonstrated that inactivation of pyroglutamyl peptidase II was not due to dissociation or internalization of this enzyme molecule. Incubation of TPA-activated Y-79 cell membranes with gamma-[32P]-ATP followed by immunoprecipitation revealed a time-dependent phosphorylation of a 48 kilodalton subunit of pyroglutamyl peptidase II. These studies indicate that the phorbol ester effect is mediated by protein kinase C, and reveal a mechanism of potentiation of the action of TRH at its target sites.

Production of insulin-like growth factor-II by human fetal pancreas in culture.

Insulin-like growth factor-II (IGF-II) is a polypeptide hormone thought to be involved in fetal development because of its wide distribution in fetal tissues and its presence in the fetal circulation. We have developed a highly sensitive radioreceptor assay for IGF-II using rat liver microsomal membranes and have used this assay to test for the presence of IGF-II in the human fetal pancreas and the release of IGF-II by the human fetal pancreas in organ culture. IGF-II was present in extracts of pancreatic tissue (0.056 +/- 0.012 pmol/mg tissue, n = 5) and was released in culture at the rate of 0.027-0.134 pmol/mg tissue per day with release being maintained for at least 3 weeks in culture. The rate of release was not affected by the gestational age of the fetus over 14 to 20 weeks but was significantly related to the rate of insulin release (r = 0.712, P less than 0.001, n = 34). Chronic exposure to 12-0-tetradecanoylphorbol-13-acetate (TPA), which inhibits insulin release in the human fetal pancreas, caused an 85% drop in IGF-II production, which was reversed when TPA was removed. These studies demonstrate that IGF-II is produced by the human fetal pancreas in a pattern similar to that of insulin. We suggest that control of IGF-II release, like that of insulin, may involve protein-kinase C and that IGF-II may have a paracrine or autocrine role in the development of fetal pancreatic function.

Phorbol ester stimulates human granulosa-luteal cell cyclic adenosine 3',5'-monophosphate and progesterone production

Human granulosa-luteal cell production of cyclic adenosine 3',5'-monophosphate (cAMP) and progesterone (P) were studied in response to 12-0-tetradecanoyl-phorbol-13-acetate (TPA). TPA specifically increased cAMP synthesis in a dose-dependent manner. A 7-fold increase occurred at a TPA concentration of 1 ng/ml. Time-course studies indicated that the increase in accumulation of cAMP into culture media became detectable at 4 h and continued up to 72 h. TPA also enhanced P synthesis, but the increase was statistically significant only at 72 h. Indomethacin prevented TPA-stimulated cAMP and P production. The results suggest that TPA stimulates granulosa-luteal cell cAMP and P production, and that the action of TPA is mediated by the increase in prostaglandin synthesis.

A Combination of a T Cell-Derived Lymphokine Differentiation-Inducing Activity and a Physiologic Concentration of Retinoic Acid Induces HL-60 to Differentiate to Cells With Functional Chemotactic Peptide Receptors

The human acute promyelocytic leukemia cell line HL-60 is induced by retinoic acid (RA) and N,N-dimethylformamide (DMF) to differentiate into cells having many of the functional and morphologic characteristics of mature granulocytes. With normal human phagocytic cells there is both superoxide anion (O2-) production and chemotaxis in response to chemoattractants such as N-formyl-methionyl-leucyl-phenylalanine (FMLP). We have now found that although HL-60 cells induced with RA alone produce O2- in response to 12-0-tetradecanoyl-phorbol-13-acetate (TPA) they are deficient in FMLP-stimulated O2- production and chemotaxis. In contrast, HL-60 induced either with DMF or with a combination of 10 nmol/L RA and a T cell-derived lymphokine, differentiation-inducing activity (DIA), produce O2- and exhibit chemotaxis in response to FMLP. The basis for these results appears to be the concentration of cell surface chemotactic peptide receptors. Thus, untreated HL-60 and HL-60 induced with either RA alone or DIA alone do not have measurable levels of FMLP receptors, whereas HL-60 induced with a combination of RA and DIA has 5,400 receptors per cell. HL-60 induced with RA and DIA plus 1 mumol/L dexamethasone have 25,000 receptors per cell and have greater chemotactic activity than HL-60 induced with the combination of RA and DIA. Thus, differentiation of HL-60 to cells with many properties of normal phagocytes can be induced in vitro by physiologic substances.

In-vitro induction of some features of hairy cell leukemia in chronic lymphocytic leukemia and immunocytoma cells.

In an attempt to induce in vitro differentiation we exposed cells from patients with chronic lymphocytic leukemia (CLL) and with immunocytoma (IC) to 12-0-tetra-decanoylphorbol-13-acetate (TPA) at 160 nM. After 3 to 5 days of culture cells became enlarged and the CLL cells developed a basophilic cytoplasm with an excentric nucleus. In some instances cells with many fine projections were seen. We then employed the monoclonal antibody (MAB) HD 6, which was generated against hairy cell leukemia (HCL) cells and reacts most strongly with such cells but is unreactive with plasmocytoma cells and with most CLL cells. TPA treatment induced a greatly enhanced HD 6 binding in CLL and IC cells compared to controls as demonstrated by indirect immunofluorescence. Cytochemical studies revealed that at the same time an acid phosphatase was induced, which was found to be tartrate-resistant in every instance tested. Thus, several features of HCL can be induced in CLL and IC demonstrating the close relatedness of these entities.

Inhibition of transferrin binding and iron uptake of hematopoietic cell lines by phorbol esters

Phorbol esters inhibit cell growth and the binding of transferrin to receptors on K 562, HL 60 and U 937 human leukemic cell lines. Exposure of these cells to 12-0-tetradecanoyl phorbol-13-acetate (TPA) at 37°C results in a 40% reduction of the specific binding of 125I-transferrin, which is apparent within 15 min. Half-maximal inhibition occurs at about 1 nM. Other tumor promoting phorbol esters also inhibit 125I-transferrin binding in a dose-dependent manner which parallels their known promoting activity in vivo. TPA reduces the number of transferrin receptors, and does not alter the degradation or the internalization of transferrin. In addition, TPA inhibits iron uptake by these cell lines. These effects are specific, since phorbol esters do not affect either cell growth or the binding of transferrin to Friend erythroleukemia cells and Raji cell line. On the basis of these findings it is suggested that the inhibition of transferrin binding may represent one of the mechanisms by which phorbol esters affect the growth and the differentiation of hematopoietic cell lines.

Generation of procoagulant activity (PCA) by phorbol-esters-induced macrophages derived from a leukemic promyelocytic cell line (HL-60)

Normal human monocytes and macrophages, as well as leukemic promyelocytes, generate potent procoagulant activity (PCA) resembling thromboplastin. In the present study only mild PCA was detected in a leukemic promyelocytic cell line (HL-60) and in promyelocytic cells induced to differentiate into neutrophils by dimethylsulfoxide (DMSO). After exposure to 12-0-tetradecanoylphorbol-13-acetate (TPA), the leukemic promyelocytic cells assume characteristics specific to monocytes and macrophages. This differentiation was associated with potent PCA generation. No PCA was found in lymphoblasts and several lymphoid cell lines after exposure to TPA. The PCA in TPA-induced HL-60 cells resembles tissue thromboplastin and normal monocyte PCA in several aspects: (A) All three accelerate clotting through the extrinsic coagulation pathway; (B) all three are inhibited by concanavalin-A and protected by methyl-α-D-mannopyranoside, suggesting that the PCA contains carbohydrate moieties; (C) the PCA is located in the cell membrane. However, the mechanism of PCA generation in normal monocytes and in TPA-induced HL-60 cells differs in several aspects: (A) Endotoxin and phytohemagglutinin, which are potent stimuli of PCA generation in normal monocytes, have no effect on TPA-induced HL-60 cells; (B) HL-60 cells treated with TPA develop PCA before acquiring any other specific characteristic of monocyte or macrophages; (C) the development of PCA in TPA-induced macrophages is not dependent on cell adhesion or the presence of T cells. In spite of these differences, the production of PCA is additional evidence of the similarity between TPA-induced macrophages and normal human monocytes and macrophages. The HL-60 cells may provide a unique model for studying PCA generation in mononuclear cells.

Stimulation of DNA synthesis by transient exposure of cell cultures to TPA or polypeptide mitogens: induction of competence or incomplete removal?

A transient exposure of cell cultures to 12-0-tetradecanoyl-phorbol-13-acetate (TPA) is sufficient to stimulate DNA synthesis during a subsequent incubation in TPA-free medium. We show that (1) a substantial fraction of TPA remains bound to cultures following a transient exposure to TPA and thorough washing, (2) the ability of TPA to induce DNA synthesis is a function of the amount of TPA bound to cell cultures irrespective of whether it is incubated continuously with cultures or transiently exposed to cultures under various conditions, and that (3) a transient exposure of cultures to phorbol-12-13-dibuytrate (PDB), a mitogenic phorbol ester which binds reversibly to cell cultures, does not stimulate DNA synthesis during a subsequent incubation in PDB-free medium. Therefore the persisting effects of TPA are due to it binding to cultures in a manner resistant to washing and not due to the induction of a stable cellular change prerequisite for mitogenesis. Further, we show that certain combinations of polypeptide growth factors induce DNA synthesis in the absence of any such stable cellular change. Evidence is also presented that the persisting effects on DNA synthesis following transient exposure of cultures to other polypeptide growth factors (e.g., platelet-derived growth factor) reflect tenacious binding rather than induction of a lasting biological event.

Initiator and promoter induced specific changes in epidermal function and biological potential.

Mouse epidermal basal cells can be selectively cultivated in medium with a calcium concentration of 0.01--9.09 mM. Terminal differentiation and sloughing of mature keratinocytes occur when the calcium concentration is increased to 1.2--1.4 mM. When basal cell cultures are exposed to chemical initiators of carcinogenesis, colonies of cells that resist calcium-induced differentiation evolve. Likewise, basal cells derived from mouse skin initiated in vivo yield foci that resist terminal differentiation. This defect in the commitment to terminal differentiation appears to be an essential change in initiated cells in skin and is also characteristic of malignant epidermal cells. This model system has also provided a means to determine if basal cells are more responsive to phorbol esters than other cells in epidermis and to explore the possibility that heterogeneity of response exists within subpopulations of basal cells. The induction of the enzyme ornithine decarboxylase (ODC) was used as a marker for responsiveness to phorbol esters. ODC induction after exposure to 12-0-tetradecanoylphorbol-13-acetate (TPA) in basal cells is enhanced 20-fold over the response of a culture population containing both differentiating and basal cells. When basal cells are induced to differentiate by increased calcium, responsiveness to TPA is lost within several hours. In basal cell cultures, two ODC responses can be distinguished. After exposure to low concentrations of TPA or to weak promoters of the phorbol ester series, ODC activity is maximal at 3 hr. With higher concentrations of TPA, the ODC maximum is at 9 hr. These results are consistent with the presence of subpopulations of basal cells with differing sensitivities to TPA. Other studies that use the enzyme epidermal transglutaminase as a marker for differentiation support this conclusion. In basal cell culture TPA exposure rapidly increases transglutaminase activity and cornified envelope development, reflecting induced differentiation in some cells. As differentiated cells are sloughed from the dish, the remaining basal cells proliferate and become resistant to induced differentiation by 1.2 mM calcium. These data provide additional evidence of basal cell heterogeneity in which TPA induces one subpopulation to differentiate while another is stimulated to proliferate and resists a differentiation signal. Tumor promoters, by their ability to produce heterogeneous responses with regard to terminal differentiation and proliferation, would cause redistribution of subpopulations of epidermal cells in skin. Cells that resist signals for terminal differentiation, such as initiated cells, would be expected to increase in number during remodeling, Clonal expansion of the initiated population could result in a benign tumor with an altered program of differentiation. In skin, benign tumors are the principal product of 2-stage carcinogenesis. Subsequent progression to malignancy may involve an additional step, probably a genetic alteration, that is independent of the tumor promoter.

Tumor promoter induces loss of anchorage dependence in human skin fibroblasts from individuals genetically predisposed to cancer.

To examine the role of germinal mutation in transformation by phorbol esters, we studied the induction of anchorage-independent variants of mutant human diploid fibroblasts derived from normal-appearing skin of individuals with hereditary adenomatosis of the colon and rectum (ACR). Liquid cultures were chronically exposed to 12-0-tetradecanoyl phorbol-13-acetate (TPA), then plated in agar and injected subcutaneously into athymic mice. Cultured ACR cells showed an unusual biphasic dose response to TPA. Colony-forming cells in agar were obtained at a frequency of about 5 x 10(-5). They did not, however, seem to increase in frequency during subsequent passages in liquid cultures continuously exposed to TPA. The isolated anchorage-transformed clones showed an altered clonal morphology and a considerable increase in cloning efficiency in liquid cultures and agar. The results suggest that ACR cells may be used to screen for potential tumor promoters in our environment.

The possibility of aggregation of ribosomal rna during hot phenol-SDS deproteinization

The phenobarbital-induced activation of cytochrome P4502H1 (CYP2H1) and 5-aminolevulinate synthase (ALAS-1) genes in chick embryo hepatocytes occurs at the level of gene transcription, but the molecular mechanism underlying this induction is not understood in detail. In the present study, we report that the protein kinase inhibitor 2-aminopurine markedly inhibits the phenobarbital-induced activation ofCYP2H1and ALAS-1 genes as measured by Northern blot analysis, but does not alter the basal expression of these genes in the absence of drug. Transient expression studies confirmed these findings. The construct pCATBg4.8 contains a 4.8-kb drug-responsive domain of theCYP2H1gene fused to the enhancerless SV40 promoter and the drug-induced expression of this construct in chick embryo hepatocytes was inhibited by 2-aminopurine. Another construct pCAT, with the first 547 bp of 5′ flanking region of theCYP2H1gene, is not responsive to drug and basal expression of this construct was not altered by the addition of 2-aminopurine. The evidence presented here demonstrates that the inhibitory action of 2-aminopurine on drug-induction is not due to a toxic effect on the cells. The induction of theCYP2H1gene by phenobarbital was not altered by treating cells with the specific inhibitors for protein kinase C (GF 109203X and Ro 31-8220) or prolonged exposure to 12-0-tetradecanoyl- phorbol 13-acetate (TPA) or treatment with the specific inhibitors for tyrosine kinase (genistein and tyrphostin A25). Overall, the data indicate that a 2-aminopurine-sensitive protein kinase activity is required for the phenobarbital-induction mechanism but this is unlikely to be a protein kinase C or tyrosine kinase. It can be postulated that phosphorylation of a drug receptor protein may be an important step in the drug-induction process.